1. Field of the Invention
The present invention belongs to the fields of protein chemistry, peptide chemistry, mass analysis and diagnostic medicine, and relates to a method of analyzing an oxidation state of a methionine residue in a protein sample.
2. Description of the Related Art
Heretofore, as a quantification method for an oxidized methionine residue, there have been employed various methods, such as a method based on alkaline hydrolysis of a protein sample, a method based on alkylation and hydrolysis of a methionine residue, and a method based on bromocyan cleavage and acid hydrolysis. In these conventional methods, methionine sulfoxide after hydrolyzation is detected and quantified by measuring UV absorption spectra using a liquid chromatography (LC) system. A method based on alkylation using iodoacetate labeled by the radioisotope carbon-14 (14C), and hydrolysis, has also been employed (see, for example, SCHACHTER H; DIXON GH, “Preferential oxidation of the methionine residue near the active site of chymotrypsin”, Journal of Biological Chemistry, March 1964, Vol. 239, pp 813-829). In this method, methionine sulfoxide after hydrolyzation is detected and quantified by radioactivity measurement.
It is reported that protein oxidation occurring in vivo causes decrease in protein activity and development of toxicity, and is deeply involved in diseases, such as cataract, lung emphysema, rheumatism, asthma, and metabolic syndrome and cardiovascular diseases including arteriosclerosis, and further in Alzheimer's disease and aging. The protein oxidation does not occur equally at any in vivo site but there are an oxidation-susceptible site and an oxidation-insusceptible site. It would have a significant meaning for a clarification of vital phenomena and structure-activity relationships to know a site and degree of protein oxidation under in vivo or in-vitro conditions.
The present invention focuses on an oxidation degree of a methionine residue. A methionine residue is an amino-acid residue susceptible to oxidation, and therefore receives great attention in researches on oxystress and aging-related changes.
However, the conventional methods are incapable of accurately quantifying an oxidized methionine residue.
For example, the above quantification methods using ultraviolet-visible spectra cannot quantify an oxidized methionine residue with acceptable accuracy. Moreover, the conventional methods are designed to determine an oxidation rate for the entire protein, and thereby it is unable to find out at what site of a protein and to what degree a methionine residue is oxidized.
As shown in FIG. 1B, in the conventional methods, a methionine residue which has not been oxidized in vivo is likely to be accidentally oxidized in a pretreatment stage to be performed in advance of a measurement operation, due to its oxidation-susceptible property. Consequently, the measurement is liable to fail to accurately reflect the in vivo oxidation state. That is, there is a problem of credibility of a measurement result.
For example, an oxidation-state measuring method may comprise preparing a target sample with an oxidation state to be measured, and a reference sample to be compared with the target sample, subjecting the two samples to a pretreatment, and comparing between respective measurement results of the two pretreated samples (i.e., two-group comparison analysis).
In this method, if a certain difference is detected in the two-group comparison analysis, it will be assumed that there is a difference between respective oxidation states of the two samples. However, such a difference is likely to be detected in the following situations involving accidental oxidation in the pretreatment stage: one situation where, when there is no difference between respective in vivo oxidation states of the two samples, accidental oxidation occurs mostly in one of the samples in the pretreatment stage, and a resulting difference is detected; and another situation where, when there is a certain difference between respective in vivo oxidation states of the two samples, accidental oxidation occurs mostly in one of the samples in the pretreatment stage, and a resulting difference changed from the difference between the in vivo oxidation states is detected.
Further, even if no difference is detected in the two-group comparison analysis, the detection result representing no difference is likely to come out in the following situation involving accidental oxidation in the pretreatment stage: a situation where, when there is a certain difference between respective in vivo oxidation states of the two samples, accidental oxidation occurs mostly in one of the samples in the pretreatment stage, and respective oxidation states of the samples incidentally become identical to each other after completion of the pretreatment.
In either situation, there is a possibility of failing to accurately maintain an in vivo oxidation state. Moreover, the above method requires preparing both a target sample and a reference sample, which leads to a problem about increase in analysis time and cost.